The main goal of this research is to determine the fate of acid hydrolases in cells. The underlying unsolved problems we intend to address are the mechanisms by which acid hydrolases are sequestered within lysosomes, the mechanisms by which they are secreted, and the means by which their distribution is regulated. This goal is essentially the same as in our first proposal. Only the model system for this has changed from human fibroblasts to Dictyostelium discoideum. To bring this system to fruition we need to further define the chemical and physical steps in the pathway to maturation and localization of its acid hydrolases, as well as to elucidate the mechanisms of environmental modulation of their fate. This is the main thrust of our planned research. The chemical events we are concerned with are proteolytic processing, core glycosylation, conversion of endo H sensitive cores to endo H resistant forms, sulfation, phosphorylation and PM-marker. We hope to establish the order and rates of these conversions to determine the intracellular location of the events and to investigate nutritional alterations of the fate of this enzyme. Additionally, we intend to explore the possibility of developing in vitro systems for study of some of the vesicle recognition phenomena implied by the findings. The strategy we are using to study these problems involves: a) Determination of precursor production relationships, rates of processing, and gross localization (secretion versus storage) of enzyme forms; b) Determination of nutritional effects on these parameters; c) Determination of the locale of the event by cell fractionation of pulse chase labeled cells in the presence and absence of protein transport inhibitors; d) Determination of nutritional effects on protein transport; e) Use of this information to develop in vitro systems for membrane-membrane recognition so the determinants of that recognition can be analyzed, and f) Examination of the results to test competing theories of enzyme sorting and sorting regulation.